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Analytical validation of genotyping assays in

the biomarker laboratory
Jennifer A Isler1,
Ole E Vesterqvist1 &
Michael E Burczynski1†
†Author
for correspondence
1Wyeth Research,
Biomarker Laboratory,
Clinical Translational
Medicine, 500 Arcola Road,
Collegeville, PA 19426, USA
Tel.: +1 484 865 2656;
Fax: +1 484 865 9370;
E-mail: mburczynski@
wyeth.com
High-throughput, whole-genome association studies conducted in various diseases and
therapeutic settings are identifying an increasing number of single nucleotide
polymorphisms that may predict patient responses and ultimately guide therapeutic
decision-making. In order to confirm the candidate genetic markers emerging from these
studies, there is a commensurate need for pharmacogenomic laboratories to design and
analytically validate targeted genotyping assays capable of rapidly querying the identified
individual single nucleotide polymorphisms of interest in large confirmatory clinical
studies. In recent years, a number of increasingly complex technologies have been applied
to the qualitative and semi-quantitative analysis of polymorphisms and mutations in DNA.
The different approaches available for targeted DNA sequence analysis are characterized
by various pros and cons that often present technology-specific challenges to the analytical
validation of these assays prior to their use in clinical studies. Several key principles in the
analytical validation of genotyping assays – including assay specificity, sensitivity,
reproducibility and accuracy – are covered in this review article, with specific attention paid
to three major end point detection technologies currently employed in targeted
genotyping analysis: matrix-assisted laser desorption ionization time-of-flight mass
spectrometry, Pyrosequencing® and Taqman®-based allelic discrimination. Thorough
assessment of the performance of genotyping assays during analytical validation, and
careful use of quality controls during sample analysis, will help strengthen the quality of
pharmacogenomic data used to ultimately confirm the validity of exploratory biomarkers
in DNA.

Keywords: allelic
discrimination, analytical
validation, genotyping assays,
MALDI-TOF-MS,
pharmacogenetics,
pharmacogenomics,
Pyrosequencing
part of
At the outset, it is important to define the dis-
tinction between the general process of bio-
marker validation, and the specific validation of
an analytical method used to measure the bio-
marker in question. The former process – the
accumulation of sufficient data to change the
status of an exploratory biomarker to one of a
probable valid or known biomarker – implies
many years of effort and multiple confirmatory
studies [1,2,101]. The latter process, which focuses
solely on characterizing the analytical perfor-
mance of a method or test used to measure a
biomarker, can be accomplished in a relatively
shorter time period and involves the assessment
of various parameters of the assay [3,4,102]. If the
pharmacogenomic test is intended to become a
marketed commercial assay (i.e., a device requir-
ing the submission of a premarket approval
application [PMA] or a 510[k] premarket noti-
fication demonstrating substantial equivalence
to the US FDA), a series of rigorous, well-
defined criteria must be met [5,102]. If a
pharmacogenomic test is intended for research
use only, the elements of analytical validation
are typically determined by the test user(s). The
analytical validation of exploratory genotyping
assays conducted during clinical drug develop-
ment to measure polymorphisms at predefined
locations in genomic DNA is the subject of the
present review article. The definitions of
selected characteristics that define the analytical
performance of targeted genotyping assays are
presented in Table 1.
Targeted genotyping methodologies
In the subsequent sections, brief overviews of three
examples of genotyping technologies are provided.
For more comprehensive overviews focusing spe-
cifically on the molecular principles by which
these and other types of genotyping assays work,
the reader is referred to excellent review articles
focused on general genotyping methodology over-
views [6–10] or specific technologies for matrix-
assisted laser desorption ionization time-of-flight
mass spectrometry (MALDI-TOF MS) [11–14],
Pyrosequencing® [15,16], Taqman®-based allelic
discrimination (also known as Taqman SNP
Genotyping Assays) [17] or other genotyping
technologies [18,19]. The purpose of providing
brief summaries of the methods here is to lay

10.2217/14622416.8.4.353 © 2007 Future Medicine Ltd ISSN 1462-2416 Pharmacogenomics (2007) 8(4), 353–368 353
REVIEW – Isler, Vesterqvist & Burczynski

Table 1. Analytical validation parameters for genotyping methods.

Parameters Definition
Specificity The ability of a method to determine specifically the genotype in the presence of potentially interfering genes
Sensitivity The range of input amounts of DNA for which a method can accurately assign genotype calls to samples
Efficiency (call rate) The percentage of samples that meet the genotype call acceptance criteria
Reproducibility The percentage of sample that yields identical genotype calls in multiple analytical runs
Accuracy The closeness of agreement of a single genotype measurement with the ‘true genotype’
Sample stability Determination of the effects of storage time and temperature on the stability of isolated genomic DNA
Quality assessment The process of establishing acceptance criteria for analytical runs using quality control samples with
known genotypes

the foundation for the similarities and
differences involved in various aspects of ana-
lytical validation of assays that employ distinct
molecular principles of DNA sequence
polymorphism detection.
A critical concept highlighted for each tech-
nology in the following sections is the concept of
how ‘confidence’ is calculated for genotyping
calls made by each method. Each platform uses
different parameters regarding the data output to
determine the confidence score of a genotyping
call. Although manufacturers may give default
recommendations, it is ultimately up to the end
user to establish the acceptance criteria for
assigning genotypes for each assay designed. The
predefined cut-offs for confidence scores estab-
lished by end users impact every analytical vali-
dation parameter described below and should
thus be carefully considered during genotyping
assay design and validation. A list of example
acceptance criteria for each platform (contrasted
with the assignment of genotyping calls by a
technique such as polymerase chain reaction
with restriction fragment length polymorphisms
[PCR-RFLP]) is presented in Table 2.
Primer extension & MALDI-TOF MS
Analysis of polymorphisms using primer exten-
sion assays coupled with MALDI-TOF MS
entails three major steps [11–14]:
• PCR amplification of the sequence containing
the polymorphic site;
• Primer extension through the polymorphic
site in the presence of a combination of deoxy-
nucleotides (dNTPs) and dideoxynucleotides
(ddNTPs);
• MALDI-TOF MS analysis of the primer-
extended products.
One version of this general procedure as
developed by Sequenom® (San Diego, CA,
USA), collectively termed the homogenous

Table 2. Data outputs, quality assessments and example acceptance criteria* for different methods
employed for targeted genotyping assays.
Genotyping platform Data format Quality assessment Acceptance criteria
MassARRAY® MALDI-TOF-MS Extended primer Software-assigned confidence At least one of two calls must be assigned
mass spectra score for each genotype call a conservative score or both calls must be
assigned a moderate score
Genotype calls cannot disagree
Allelic discrimination Cluster plot of Software-assigned quality Both calls must be assigned a quality value
fluorescence values value for each genotype call greater than 95
Genotype calls cannot disagree
Pyrosequencing® Pyrogram of Software-assigned confidence Both calls must be assigned a ‘pass’ or
luciferase intensities score for each genotype call ‘check’ confidence score
‘Check’ scores must be visually confirmed
Genotype calls cannot disagree
PCR-RFLP Agarose gel DNA banding pattern DNA bands must match an expected
banding pattern
*The genotype call acceptance criteria listed above are nonstringent recommendations based on the performance of most assays. These criteria can
be modified if deemed necessary to increase the call rate of otherwise accurate and reproducible genotyping assays.
PCR-RFLP: Polymerase chain reaction with restriction fragment length polymorphisms; MALDI-TOF-MS: Matrix-assisted laser desorption ionization
time-of-flight mass spectrometry.

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 Analytical validation of genotyping assays in the biomarker laboratory – REVIEW

MassEXTEND® (hME) assay, is presented in
Figure 1. After amplifying a region of genomic
DNA containing the polymorphic site of inter-
est, shrimp alkaline phosphatase is added to
dephosphorylate residual nucleotides from the
PCR reaction prior to initiating the primer
extension reaction. In the next step an extension
reaction employs a primer that anneals to the
PCR amplicon and is located with its 3´-end
juxtaposed to the polymorphic site. Addition of
a ‘termination mix’ (containing a specified com-
bination of nonterminating dNTPs and chain-
terminating ddNTPs) to initiate the primer
extension reaction causes dNTPs to be incorpo-
rated until a sequence-dependent ddNTP incor-
poration event terminates the reaction. The
assay is designed to allow immediate termina-
tion at the juxtaposing polymorphic site for one
of the alleles possessing a nucleotide comple-
mentary to the chain-terminating ddNTP, and
termination further downstream for the other
allele. A typical reaction generates allele-specific
primer extension products that are generally
1–4 bases longer than the original primer. Since
the termination point and number of nucleo-
tides incorporated are sequence-specific, the
mass of the extension products can be used to
identify the nucleotide present at the poly-
morphic site. Sequenom has more recently
developed an iPLEX® assay, a modified hME
assay that uses a common termination mix with
mass-modified terminators. One significant dif-
ference is that all primer extension reactions ter-
minate after a single base extension, allowing for
increased plexing efficiency [103].
Following a critical resin-based purification
step to eliminate salts present in previous reac-
tion buffers, a minute quantity of the reaction
(∼15 nl) is transferred to a solid matrix for mass
determination by MALDI-TOF MS. Primer
mass spectra data can be directly converted into
genotype calls using software such as Spec-
tro-TYPER™ (Sequenom), in which prespeci-
fied single peak masses or peak mass patterns
represent various allelic combinations. In our
laboratory, samples are analyzed in duplicate, and

Figure 1. Molecular basis for primer extension/matrix-assisted laser desorption

ionization time-of-flight mass spectrometry assays.

Allele 1 Allele 2

EXTEND primer (23-mer) EXTEND primer (23-mer)

TCT ACT

+ enzyme
+ ddATP
Extended primer (24-mer) + dCTP/dGTP/dTTP
Extended primer (26-mer)

TCT ACT

21 22 23 24 25 26 27 28 29 21 22 23 24 25 26 27 28 29
Allele 1

Allele 2
EXTEND primer
EXTEND primer
Allele 1

5000 6000 7000 8000 9000 10,000 5000 6000 7000 8000 9000 10,000

Detection of differentially extended primer extension products by MALDI-TOF MS analysis. As indicated in

the text, primers are annealed upstream of polymorphic sites and dideoxynucleotide termination mixes
enable differential extension through wild-type versus polymorphic alleles. The different-sized products
relate to the identity of the nucleotide at the polymorphic position and the different masses in the TOF
spectra exhibit a direct correlation with the different alleles of interest.
dCTP/dGTP/dTTP: Deoxynucleotides; ddATP: Dideoxy adenosine triphosphate;
MALDI-TOF MS: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Reproduced from [11] with permission of Future Drugs Ltd.

future science group www.futuremedicine.com 355

REVIEW – Isler, Vesterqvist & Burczynski

genotype calls are issued only if genotype calls for
the duplicates are of high confidence and in
agreement. The degree of confidence assigned to
genotyping calls using this system depends upon
peak characteristics and is mainly determined by
the closeness of the observed atomic masses with
the expected masses of primer extension products
in the sample, and the signal-to-noise ratios
observed for each of the peaks. As mentioned
previously, during analytical validation of any
genotyping assay using primer exten-
sion/MALDI-TOF analysis, it is critical to deter-
mine confidence score acceptability criteria for
genotype assignments prior to running clinical
samples. An example of the raw spectra generated
from a multiplexed MALDI-TOF assay in which
three polymorphic positions were interrogated in
a single assay is shown in Figure 2.
Pyrosequencing
Pyrosequencing, as developed by Biotage (Upp-
sala, Sweden), differs from other methods in that
it provides genotyping results in the context of
the neighboring DNA sequence [15,16]. In this
assay (depicted in Figure 3) a sequencing primer is
hybridized to a single stranded template isolated
from a PCR reaction using streptavidin beads
following PCR amplification of the target DNA
sequence. In the first of four enzymatic reactions,
dNTPs are added in a predetermined order and
DNA polymerase catalyzes the incorporation of
each dNTP complementary to the base present
in the DNA template. Each incorporation event
is accompanied by release of pyrophosphate in
an amount proportional to the amount of dNTP
incorporated. ATP sulfurylase then converts
pyrophosphate into ATP, which drives luciferase-
mediated conversion of luciferin to oxyluciferin
and generates visible light in amounts propor-
tional to the amount of ATP. The light emitted is
detected by a camera and visualized as a peak in a
pyrogram. Apyrase is added to degrade unincor-
porated dNTPs and excess ATP prior to addition
of the next dNTP, allowing the complementary
DNA strand to be synthesized and the nucle-
otide sequence to be determined from the signal
peaks in the resulting pyrogram.
Computer software such as PyroMark MD
1.0™ (Biotage) can be used to determine the
optimal dNTP dispensation order based on the
sequence surrounding the single nucleotide
polymorphism (SNP) of interest, and the same
software can generate all theoretical pyrograms
as bar graphs. The software then calculates peak
heights from the raw data, and genotypes are
determined by comparison with the theoretical
pyrograms. Genotype calls are assigned quality
scores of pass, check or fail based on a number of

Figure 2. Representative data outputs from primer extension/MALDI-TOF MS assay.

UEP CYP2D621584
UEP CYP2D641846

UEP CYPD632549

Pausing peak
Pausing peak
Pausing peak

M.DEL
M.DEL

DEL
CG

AG

CG
AG

G
C
M

C
A

40
Intensity (signal)

30

20

10

4800 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800

Mass (amu)
Mass-spectral image from a multiplexed primer extension/MALDI-TOF MS assay. Dotted lines represent either
unextended primers (UEP), potential pause/early termination peaks, or successfully extended primers
indicative of the presence of different alleles. The assays shown are for CYP2D6*2 (pink), CYP2D6*3 (blue)
and CYP2D6*4 (green). This sample was determined to be wild-type with respect to all three alleles, as
shown by only a single peak (indicated by arrows) at the location of each wild-type extended primer.
CYP: Cytochrome P450; MALDI-TOF-MS: Matrix-assisted laser desorption ionization time-of-flight
mass spectrometry.

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 Analytical validation of genotyping assays in the biomarker laboratory – REVIEW
Figure 3. Molecular basis for Pyrosequencing® assays.
Nucleotide-incorporation-dependent detection of light by the Pyrosequencing
assay. In this multi-enzymatic cascade, light is emitted by the dual actions of
sulfurase and luciferase only if the polymerase catalyzes nucleotide addition to
the growing strand upon dispensation of one of four nucleotides into the
reaction. A predetermined dispensation order is used to dispense nucleotides
into the reaction, and the pattern of light emission corresponds to the sequence
of the target gene interrogated. Apyrase is added at the conclusion of every
dispensation to degrade the previously added nucleotide before the next
nucleotide is dispensed.
PPi: Pyrophosphate.
Reproduced from [16] with permission of Future Drugs Ltd.
factors, including the agreement between the
theoretical and actual pyrograms, observed sig-
nal:noise ratios and calculated peak widths.
Genotype calls are typically only accepted if
assigned a pass score, or if assigned a check score
and confirmed visually by the user. An example
of a theoretical pyrogram and the actual pyro-
gram for a sample bearing the genotype pre-
dicted from the theoretical pyrogram below it
are presented in Figure 4.
Taqman 5´-nuclease-based
allelic discrimination
Taqman-based allelic discrimination (referred to
hereafter as simply allelic discrimination), using
Taqman SNP Genotyping Assays (Applied Bio-
systems, Foster City, CA, USA), provides another
method for polymorphism detection (Figure 5).
This assay utilizes a standard pair of PCR primers
to amplify a region containing the polymorphism
of interest, and two detection probes [17]. The two
detection probes bind differentially to amplified
products containing either the nonpolymorphic
or the polymorphic nucleotide, each of which is
labeled with a different fluorescent dye on its
future science group www.futuremedicine.com
5´-end and a quencher on its 3´-end. During
PCR, each detection probe anneals specifically to
its complementary sequence within the PCR-
amplified region. As the product is amplified, the
5´-nuclease activity of DNA polymerase releases
the reporter dye from its proximity to the
quencher allowing fluorescence. When the probe
is intact, the proximity of the reporter dye to the
quencher dye results in suppression of the reporter
fluorescence. However, when hybridized to its tar-
get, the probe is cleaved by DNA polymerase and
the reporter and quencher dyes are separated,
resulting in an emission of a fluorescent signal.
At the conclusion of the assay, the accumulated
fluorescence levels of both probes in each sample
are measured. ABI software plots the fluorescence
values for each detection probe in each sample
using a 2D cluster plot in which the 2D location
of each sample is determined by the strength of
fluorescence for each of the detection probes, and
relates, therefore, to the identity of the alleles
originally present in the sample. An algorithm
assigns a confidence score (referred to as a ‘quality
value’) for each genotype call based upon the
‘closeness’ of each sample with other samples
exhibiting similar fluorescence properties.
Homozygotes for one allele have increased fluo-
rescence in one channel but baseline fluorescence
in the other, while heterozygotes possessing both
alleles exhibit intermediate fluorescence in both
channels. The quality value ranges from 0–100,
and the default minimum quality value required
for an acceptable genotype call in an allelic dis-
crimination assay is set to 95. However, once the
accuracy of a genotyping assay employing allelic
discrimination is established, the stringency for
these quality values can be either increased or
decreased as appropriate. Figure 6 shows an exam-
ple of a cluster plot displaying samples
homozygous for allele X (red), allele Y (blue) or
heterozygous for both (green).
Comparison of
genotyping methodologies
All of the technologies presented above have per-
ceived advantages and disadvantages, and these
are listed in Table 3. PCR-RFLP is also included
for comparison. The following sections highlight
a few of the most relevant pros and cons of the
technologies covered in this review. Optimal
technologies to use are almost always assay-
dependent, but often any of these or other tech-
nologies can be used to generate genotyping data
of sufficient quality after careful assay validation
if one is limited to one type of platform or
357
REVIEW – Isler, Vesterqvist & Burczynski

Figure 4. Representative data outputs from a Pyrosequencing® assay.

A
C/C G/G

Relative amount
2.0

1.0

0.0
G T C T A G C T G C T A
Nucleotide dispensation order
B
1600
Intensity (luminescence)

1500

1400

1300

E S G T C
A GT C T G C T A
5 10
Nucleotide dispensation order
A theoretical pyrogram (A) and the actual pyrogram (B) are presented for an assay in which two successive
polymorphic regions were queried (highlighted in purple) and indicated in the following sequence:
[T/C]A[C/G]CGCTA. In a typical Pyrosequencing assay, the light emission prior to reagent addition is captured
(E), the light emission after addition of reagents is captured (S) and, finally, the light emission after addition
of an initial nucleotide that should not be present as the next nucleotide is captured (in this case, G). After
these control steps, the possible nucleotides at the first polymorphic position are added sequentially (in this
case, T followed by C), then a nucleotide that should not be present as the next nucleotide (in this case, T)
followed by the next nonpolymorphic nucleotide in the sequence (A) and, finally, the next two possible
nucleotides in the second polymorphic region (in this case, G followed by C).

another. The comparisons below are made only to
highlight the performance differences between
these platforms and to draw attention to a core set
of considerations before developing genotyping
assays on any platform.
Multiplexing capabilities
Primer extension assays employing
MALDI-TOF MS offer a high-throughput
method capable of high-order multiplexing of
SNP assays, since a large number of extension
primers and extended primer products of vary-
ing length and composition can be resolved and
detected within the range of a typical TOF mass
spectrometer. While the possibilities for multi-
plexing with MALDI-TOF MS are not infinite,
since all primers and extension products must be
adequately resolved, multiplexing of up to
24 alleles has been reported [103]; however, both
Pyrosequencing and allelic discrimination have
lower multiplexing capabilities. Pyrosequencing
can be multiplexed into duplex and triplex
assays, but these require careful assay design and
software-assisted interpretation. Multiplexing of
Taqman-based allelic discrimination assays is
currently limited by the wavelengths of the fluo-
rophores that can be simultaneously measured
during real-time PCR.
Sensitivity & susceptibility
to contamination
Sensitivity is a double-edged sword with respect
to PCR-based genotyping assay performance.
High sensitivity is critical when very small quanti-
ties of DNA are available (e.g., forensic samples),
but is not necessarily as critical when ample
amounts of DNA are available in the samples of
interest (5 ml whole blood draws from subjects
participating in clinical studies, for example). In
our experience, both MALDI-TOF and

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 Analytical validation of genotyping assays in the biomarker laboratory – REVIEW

Figure 5. Molecular basis for Taqman®-based allelic discrimination assays.

MGB
Forward R Q
primer Probe
5´ 3´
3´ 5´

5´ 3´ Legend
5´
Reverse
primer VIC
Allele 1 FAM
F
V Quencher
MGB MGB

Q Reporter
Q
Minor groove
binder
Match Mismatch Ampli Taq
Allele 2 Gold™ DNA
polymerase
F F
MGB MGB

Q Q

Match Mismatch

Allele-specific oligonucleotide hybridization-dependent fluorescence emission by a Taqman® allelic

discrimination assay. In real-time PCR-based allelic discrimination assays, two detection probes (containing a
fluorophore and a quencher at each end) are used that differ only in the fluorophore attached and the
identity of the central nucleotide. Each detection probe is carefully optimized to hybridize to either the
wild-type or polymorphic sequence of interest, but not both, under the recommended conditions for PCR
amplification. Only when a probe is hybridized to a perfect match will it become hydrolyzed during PCR,
allowing detection of the fluorophore after release from its proximity to the quencher molecule.
F: FAM; FAM: Fluorescent dye, homozygous for allele 2; MGB: Minor groove binder; PCR: Polymerase chain
reaction; Q: Quencher; R: Reporter; V: VIC; VIC: Fluorescent dye, homozygous for allele 1.
Reprinted from [17] with permission of Elsevier.

Pyrosequencing provide extremely high levels of Throughput

sensitivity and can, in fact, be used to detect
minor fractions of a polymorphism within a large
heterogeneous pool. As alluded to before,
extremely high sensitivity can actually lead to
problems via detecting spurious signals in nega-
tive controls due to minute levels of contamina-
tion during sample preparation. We have
observed this problem, albeit infrequently, with
all three technologies, which is the basis for the
quality control (QC) requirement that analytical
results be accepted only in the presence of ‘clean’
no template controls during any analytical run.
To minimize the incidence of contamination and
false positives in genotyping assays, genotyping
laboratories can employ uracil N-glycosylase anti-
contamination technology to prevent the carryover
of former PCR products into new reactions [20].
Allelic discrimination and MALDI-TOF-based
genotyping assays currently offer 384-well
throughput, while Pyrosequencing currently
offers 96-well throughput. While all three tech-
nologies utilize PCR as an initial step, Taqman-
based allelic discrimination requires the least
amount of time to completion since the PCR
and probe hybridization steps occur simultane-
ously. Both Pyrosequencing and MALDI-TOF
require additional hands-on steps following PCR
amplification. Pyrosequencing requires a brief
sequence of strand denaturation, primer hybridi-
zation and strand elongation, whereas MALDI-
TOF is the most labor-intensive and requires
two additional enzymatic reactions followed by a
resin-based cleanup (to eliminate the effect of
positive ions during MALDI-TOF analysis) and

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REVIEW – Isler, Vesterqvist & Burczynski

Figure 6. Representative data outputs from primer extension/MALDI-TOF-MS,

Pyrosequencing® and allelic discrimination assays.

Fluorescence emission by a group of samples analyzed in an allelic discrimination assay. Samples exhibiting
fluorescence in only one channel (over baseline) indicate the presence of only one allele (blue and red
samples) and are homozygous for one allele or the other. Samples exhibiting fluorescence in both channels
indicate the presence of both alleles and are heterozygotes at that position.
CYP: Cytochrome P450; MALDI-TOF MS: Matrix-assisted laser desorption ionization time-of-flight mass
spectrometry; NTC: Non-template control.

chip spotting. For 96 samples, the average assay
time from initial reaction set-up to data output
for allelic discrimination assays is approximately
2 h; for Pyrosequencing it is approximately 3 h;
and for MALDI-TOF it is approximately 5 h.
The same times hold true for 384 samples for
allelic discrimination and MALDI-TOF, but
increase significantly for Pyrosequencing in the
current 96-well format. The use of automated
liquid handling and accessories that enable assay
plates to be stacked for continuous analysis can
increase throughput even further.
Sequence context
Only Pyrosequencing affords the user an
opportunity to generate true sequence context
around the SNP of interest, since Pyrosequenc-
ing reconstructs the sequence downstream of
the sequencing primer based upon light emis-
sion after single nucleotide addition in every
sample analyzed. This is an attractive quality
since, when used properly, it can provide an
‘internal standard’-like assessment of the assay
specificity for every sample (see next section).
While it is unlikely that MALDI-TOF-based
primer extension assays could yield primer
extension masses falsely identified as genotype
calls after nonspecific hybridization to a target
gene, it is nonetheless a possible scenario, espe-
cially for pseudogenes. Allelic discrimination
assays only generate a fluorescence signal at the
end point of allele detection, and the chance

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 Analytical validation of genotyping assays in the biomarker laboratory – REVIEW
Table 3. Select advantages/disadvantages of polymerase chain reaction with restriction fragment length
polymorphisms versus targeted genotyping technologies in present review.
Platform Advantages
PCR-RFLP Low cost
Simple instrumentation
MALDI-TOF MS High throughput (384-well)
High signal to noise
Mass accuracy
Multiplexing capabilities
Excellent sensitivity
Pyrosequencing® Moderate throughput (96-well)
Sequence context for every sample
Good for insertions and deletions
Allelic discrimination High throughput (384-well)
Common instrumentation in many laboratories
PCR-RFLP: Polymerase chain reaction with restriction fragment length polymorphisms; MALDI-TOF MS: Matrix-assisted laser desorption ionization
time-of-flight mass spectrometry.
that such a signal could be due to nonspecific
binding is always theoretically present. How-
ever, these assays have been carefully optimized,
and the use of recommended PCR cycling con-
ditions should ensure sufficient specificity of
Taqman-based allelic discrimination assays.
Careful analytical validation of a genotyping
method employing any of these end point
detection technologies is the best way to dem-
onstrate to an end-user’s satisfaction the speci-
ficity of a given assay during validation, but
only Pyrosequencing can generate a specificity
measure (sequence context) for each sample
analyzed during an analytical run. Even pyro-
sequencing can miss nonspecific hybridization,
again in the case of pseudogenes, if there is
100% identity in the regions flanking the SNP
of interest.
Detection of genetic alterations other
than SNPs
All three technologies can interrogate SNPs.
Short insertions and/or deletions can be effec-
tively assessed by all three technologies as well,
but longer insertions and deletions, or nucleo-
tide expansion repeats, can be increasingly diffi-
cult to assay. Long nucleotide expansions
differing by only a few repeats can be difficult to
detect by allelic discrimination since the detec-
tion probe can hybridize nonspecifically to vari-
ous sections of the expanded repeat. Similarly,
extension primers used in MALDI-TOF assays
need to lie near or juxtapose the polymorphic
site of interest, and if the polymorphism of
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Disadvantages
Data quality
Subjective visual interpretation
Low throughput
Instrumentation cost/complexity
Susceptible to contamination due to high sensitivity
Moderate throughput (96-well)
Not amenable to higher order multiplexing
Less certainty in genotype assignments
Fluorescence as output
Low multiplexing capabilities
interest is bracketed on both sides by extensive
repeating sequences, it is impossible to direct
the primer extension primer to the
polymorphic site. In these cases, Pyro-
sequencing may be of use since the sequencing
primer can be designed to hybridize further
from the polymorphic site in a nonrepeat por-
tion of the sequence and then read through the
expansion to determine the exact number of
triplet repeats or the identity of a poly-
morphism within the expanded repeat region.
Currently this approach has an upper limit of
100 or so nucleotides under optimized condi-
tions. While long expanded repeats are less
common than typical single-point mutations
or single nucleotide polymorphisms of interest,
these are important considerations for scien-
tists interested in characterizing these types of
genetic modifications.
Instrumentation, analytical interpretation
& range of applications
Taqman-based allelic discrimination and Pyro-
sequencing both can be performed on instrument
packages that cost less than US$100,000, while
MALDI-TOF instrumentation can cost substan-
tially more. Allelic discrimination assays use the
same familiar real-time detection system from
Applied Biosystems employed for RNA quantifi-
cation by real-time PCR, and thus can be run on
a common instrumentation system in biomedical
laboratories that may not necessarily routinely
focus on genotyping assessments. Software pack-
ages provided with all three systems are
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REVIEW – Isler, Vesterqvist & Burczynski
user-friendly and all three platforms enable auto-
mated final output of genotype calls based on the
generated signal data. Each of the methods may
require visual inspection for certain outlier sam-
ples where chemical impurities or other problems
hamper the generation of easily interpretable
spectra or plots. While this rarely occurs, peak-
based outputs from Pyrosequencing and
MALDI-TOF are more objectively interpretable
than the cluster plots generated by fluorescence
intensity accumulated during allelic discrimina-
tion assays. All of the platforms have multiple
uses – for instance, the real-time detection system
used for allelic discrimination and MALDI-TOF
can also be used for gene-expression analysis, and
Pyrosequencing and MALDI-TOF can be used
for methylation assays.
Analytical validation of
genotyping assays
Pharmacogenomic information is now provided
in a growing number of new drug applications
[1,2,10,21,22,101]. Owing to the increasing impor-
tance of pharmacogenomic tests during the
development of new drug candidates, the US
FDA has issued multiple publications regarding
both general pharmacogenomic biomarker vali-
dation and principles of analytical validation of
pharmacogenomic tests. Examples of the former
include the Guidance for Industry regarding
pharmacogenomic data submissions issued in
March 2005 [101], a process map proposal for
the validation of genomic biomarkers in both
preclinical and clinical drug development [1] and
a review of the clinical utility of pharmaco-
genomic tests for drug-metabolizing enzymes
[2]. The US FDA guidance document essentially
draws a distinction between more or less well-
established pharmacogenomic biomarkers,
where genotypes that are strongly linked to cer-
tain phenotypic effects are considered either
probable or known valid biomarkers and the
other, less well-established genotypes that may
be linked to certain phenotypic effects are con-
sidered observational or exploratory biomarkers.
While both the US FDA guidance and the pro-
posal map laid out by Goodsaid and Frueh pro-
vide excellent overviews of the general
validation process for pharmacogenomic biom-
arkers (i.e., the process of converting the status
of exploratory biomarkers to that of probable or
known valid biomarker), they do not address
the more specific topic concerning the analytical
validation of pharmacogenomic tests used to
measure the genomic biomarkers in question.
362 Pharmacogenomics (2007) 8(4)
This more specific aspect is addressed in detail
in another Guidance for Industry, entitled Class II
Special Controls Guidance Document: Drug Metab-
olizing Enzyme Genotyping Systems [102]. While this
document mainly focuses on the more extensive
steps that diagnostic assay manufacturers should
follow when submitting a PMA or a 510(k) pre-
market notification for a genotyping device to the
US FDA, many of the principles outlined for spe-
cifically characterizing the test methodology are
pertinent to the topic of this review.
In the next section we describe procedures that
can be implemented to analytically validate assays
used to determine SNP genotypes in clinical trials
using three different technology platforms. Prepa-
ration of, and adherence to, standard operating
procedures outlining the analytical validation of
genotyping assays in a biomarker laboratory is
highly desirable and recommended. The main
objective in the analytical validation of any bio-
marker assay (pharmacogenomic or other) is to
characterize and control sources of variability in a
method. For a genotyping assay, this process can,
in turn, lead to an understanding of the expected
error rate [23]. While SNP genotyping methods are
typically qualitative in nature, newer technologies
can allow oncology-relevant mutation or copy
number assays to be more quantitative if needed.
Thus, key elements in the analytical validation of
most SNP genotyping methods are the evaluation
of the specificity, sensitivity, accuracy and repro-
ducibility of the methods as summarized in
Table 1. Of these, the determination of the abso-
lute accuracy of a genotyping method can be very
challenging, since well-characterized reference
materials/standards or reference methods are usu-
ally not available, although there are impressive
current efforts in this area to eventually make
standard QC samples available for genotyping
assays [24]. In most cases, accuracy for an explor-
atory SNP is best determined with minimized risk
by assuming that the convergence of genotype
calls between multiple methods employing differ-
ent molecular principles of sequence detection
indicates a ‘true’ genotyping result. Often this is
achieved by comparing the results of the assay
undergoing validation with sequence generated by
an independent dideoxysequencing reaction (see
section on Accuracy below), but any previously
validated allele-resolving assay could also theoreti-
cally be used to determine the convergence of the
genotype calls between methods.
In addition to the concepts of analytical
method validation, we also summarize recom-
mendations regarding the use of quality controls
future science group
 Analytical validation of genotyping assays in the biomarker laboratory – REVIEW
during the genotyping analysis of clinical sam-
ples. To control for unforeseen changes in
method performance, biomarker laboratories
should use QC samples to assess the quality of
data delivered in any given analytical run. The
QC samples should, whenever possible, be based
on the true sample matrix, and standard accep-
tance criteria should be prospectively defined
prior to the analysis of clinical samples. For
genotyping assays, QC samples are typically
comprised of positive controls bearing each of
the potential genotypes of interest (when possi-
ble/practical) to ensure that the method is capa-
ble of detecting all allele combinations
accurately and reproducibly. Genotyping assays
must also contain negative controls demonstrat-
ing the lack of spurious genotype calls in DNA-
free samples, which indicate the absence of con-
taminating genomic DNA that could interfere
with the assay.
Specificity
The specificity of a genotyping assay relates to
the ability of the assay to recognize and accu-
rately detect the specified polymorphism with-
out reacting with or detecting related DNA
sequences that can interfere with the assay and
confuse final genotype assignments. As such,
the specificity of any genotyping method is
highly dependent on the specificity of the prim-
ers used in the various PCR-based steps of the
reaction. This aspect of analytical validation is
particularly important for genotyping assays
designed to detect polymorphisms within genes
that have high homology to other regions in the
genome (e.g., cytochrome P450 [CYP]2D6 and
its pseudogenes CYP2D7 and CYP2D8). In
such cases, nested PCR strategies may be
required in order to first amplify the gene of
interest, and then design a second PCR-based
strategy to detect the SNP of interest in the
enriched target gene PCR product. Even then,
great care must be exercised to demonstrate that
pseudogenes originally present in the initial
genomic DNA sample will not contribute a sig-
nal in a genotyping assay employing a nested
PCR approach, despite being present at far
lower levels than the amplified target gene.
With the sequencing of the human genome,
the specificity of any nucleic acid-based assay can
be evaluated in silico during the process of PCR
primer design. For commercially available Taq-
man assays where primer and probe sequences
are proprietary, the demonstration of the speci-
ficity of PCR primers and detection probes is
future science group www.futuremedicine.com
conducted in silico by Applied Biosystems as
described in their white paper on the design of
Taqman probe-based assays [104].
For the other types of non-Taqman geno-
typing assays designed within the biomarker lab-
oratory, similar in silico analyses can be
performed. In these cases an assay can prelimi-
narily be considered specific if bioinformatic
sequence analysis (using the current Ensembl
build of the human genome) indicates that only
the target gene of interest will be amplified by
the PCR strategy. During the in silico analysis of
genotyping assay specificity, suitable binding
sites for PCR primers designed to amplify the
target SNP of interest are evaluated with respect
to whether other polymorphisms exist in the
proposed PCR primer binding sites, and
whether the PCR primer binding sites are
unique to the gene of interest or also exhibit high
homology elsewhere in the genome. Assays pre-
dicted to amplify DNA sequences other than the
target of interest must be redesigned.
Wet laboratory or experimental demonstra-
tion of assay specificity can also be performed,
for example, by sequencing the amplicon gener-
ated by the designed PCR assay to demonstrate
that the amplified sequence matches only the
target sequence intended for amplification.
Although the PCR product obtained from a
newly designed genotyping assay can be submit-
ted for direct sequencing, a more rigorous
method is to clone the PCR product and
sequence a large number of clones to determine
the percentage abundance of the target ampli-
con amongst all amplified sequences. Alterna-
tively, the specificity of a PCR-based genotyping
assay can be assessed by melting-curve analysis
of the amplified product to demonstrate the
presence of a single melting-point temperature
(Tm) in the melting curve of the product. How-
ever, such wet-laboratory-based approaches still
represent inexact determinations (for instance,
amplified products from a pseudogene can yield
a Tm indistinguishable from that of the target
gene if the amplicons share high homology),
and these experiments can also be costly and/or
time-consuming to execute. In our experience
we have found that careful in silico analysis of
PCR primer specificity often provides a suffi-
ciently accurate assessment of the overall speci-
ficity of a genotyping assay for the purposes of
analytical validation.
As a final note of caution regarding assay
specificity, since PCR can detect a few mole-
cules of the target sequence, and the end point
363
REVIEW – Isler, Vesterqvist & Burczynski
technologies in current use display impressive
sensitivity (see next section), a small amount of
contamination in a sample can lead to a mis-
leading result. It is therefore of utmost impor-
tance that the laboratory uses procedures
(e.g., automation and regular fluidics mainte-
nance) that will minimize the risk of contami-
nation by amplicons or genomic DNA from
neighboring samples, and employ QCs that will
indicate the presence of contamination during
an analytical run.
Sensitivity
Sensitivity of targeted genotyping assays may
sometimes be considered a less critical issue in
the analytical validation of pharmacogenomic
tests employed during clinical development
since:
• Milliliter volumes of whole blood are typically
drawn from subjects providing pharmaco-
genomic samples in clinical trials;
• Microgram amounts of DNA are available
from such volumes, even though genotyping
assays typically require amounts of input
DNA at the ng level.
Regardless of these considerations, however,
establishing the sensitivity of genotyping assays
provides valuable information regarding the
minimal recommended input amount of DNA
for a given method. To establish the sensitivity
of a qualitative genotyping assay, genotyping
calls are simply attempted in increasingly
diluted genomic DNA samples until the geno-
typing assay employed is no longer able to make
a genotyping call with acceptable confidence.
Establishing assay sensitivity is an especially
critical aspect of analytical validation for semi-
quantitative genotyping assays designed to
detect mutations in oncology tumor tissues that
may be genetically heterogeneous. For estab-
lishing the sensitivity of these types of assays,
genomic DNA titration experiments can be
performed, in which genomic DNA from a cell
line bearing the mutation of interest is titrated
in a decreasing relative ratio to genomic DNA
from a wild-type cell line. Similar to a standard
genotyping sensitivity experiment, the point at
which the genotyping assay is no longer able to
detect the presence of the mutation with
acceptable confidence is defined as the limit of
detection of the assay.
For a typical dideoxy-sequencing reaction, the
limit of detection of a mutation in a background
of wild-type genomic DNA is approximately
364 Pharmacogenomics (2007) 8(4)
15–20%. We have recently determined that a
primer extension/MALDI-TOF reaction can
detect as little as 1–5% mutation in a back-
ground of wild-type genomic DNA. These
results indicate that newer technologies will
afford greater sensitivity for mutation detection,
which should eventually result in commensu-
rately earlier diagnosis/prognosis than has been
available with older sequencing methodologies.
Efficiency
The efficiency of a genotyping assay (also
expressed as the overall call rate) simply indicates
the ability of a qualitative assay to provide an
analytically acceptable genotyping result (which
may either be correct or incorrect – this aspect of
analytical performance is captured by accuracy as
described below). Analytical acceptability, in this
case, is established by prospectively defining the
cut-offs for confidence scores that will allow a
genotyping call to be made when using the rec-
ommended starting input amount of genomic
DNA for analysis. To evaluate the efficiency of a
given genotyping assay, the assay should be per-
formed on a number of different days using a
validation sample panel. Whenever possible, the
validation sample panel should include each
allele combination and have an equal distribu-
tion of the genotypes or a distribution similar to
the genotype frequency in the relevant popula-
tion. To determine the efficiency of the method,
we typically perform at least three analytical runs
in an analyst-blinded fashion using at least
40 genomic DNA samples per run. The effi-
ciency or call-rate of the assay is calculated by
dividing the number of samples yielding accept-
able genotype calls by the total number of sam-
ples analyzed. Assay efficiencies of robust assays
are typically 99–100%.
Reproducibility
Reproducibility refers to the ability of a qualita-
tive genotyping assay to yield identical results
for a given sample in different analytical runs.
To evaluate the reproducibility of a given geno-
typing assay, the assay should be performed on
a number of different days using the previously
described validation sample panel. We typically
establish the overall call-rate and the reproduc-
ibility of the genotyping method at the same
time over multiple analytical runs; thus, in
order to determine the reproducibility of a
method, we typically analyze the same
40 genomic DNA samples over three or more
analytical runs. The reproducibility of the
future science group
 Analytical validation of genotyping assays in the biomarker laboratory – REVIEW
method is calculated by dividing the number of
samples yielding identical genotype calls in all
analytical runs by the total number of samples
analyzed. Again, assay reproducibility of robust
assays typically ranges from 99–100%. While
genotyping methodologies of optimized assays
are typically very reproducible and rarely yield
disagreement between replicates, measurement
of the assay reproducibility during analytical
validation also evaluates the preanalytical vari-
ables associated with the assay, including the
sample handling steps. While very rare, in our
experience most disagreements between repli-
cate samples are typically due to either variable
PCR failures or pipetting errors. To minimize
the latter source of error, the use of automated
liquid handling systems in as many individual
steps as possible is highly desirable.
Accuracy
Establishing the accuracy of a genotyping assay
is quite challenging, and this parameter actually
refers to establishing the ability of the geno-
typing assay to yield the ‘true’ genotype call.
Accuracy can be determined by comparing the
genotype call determined by a given genotyping
assay with the actual genotype of each valida-
tion sample. The actual genotype of each vali-
dation sample is most often determined using
DNA dideoxy-sequencing analysis, which is
considered the gold standard comparator
method in the field of genotyping [2,101]. How-
ever, other alternative methods of demonstrated
validity may also be used to establish actual
genotypes for the purpose of accuracy determi-
nation. An important pitfall that must be
avoided when determining accuracy is using the
same PCR strategy to amplify the target gene
for both end point detection methods. To
establish the accuracy of a genotyping assay
undergoing validation, a unique and indepen-
dent target gene PCR amplification strategy
should be used to amplify the region of
genomic DNA for sequence analysis by a sec-
ond method. Convergence between indepen-
dently designed PCR-based methods is the only
way to establish accuracy of a genotyping assay,
since convergence of two technologies for geno-
type assignments on the same PCR template
only demonstrates that the two methods pro-
vide similar results for that amplicon (the
design of which may be flawed).
We typically determine the accuracy of each
genotyping assay validated in the biomarker lab-
oratory by submitting the 40 genomic samples
future science group www.futuremedicine.com
analyzed for efficiency and reproducibility to
analysis by DNA sequencing. Alternatively,
when there is a well-characterized/published
method available, (e.g., the US FDA-approved
Amplichip® CYP450 test for CYP2D6 or
CYP2C19 [Roche Diagnostics, Indianapolis, IN,
USA]), a method comparison study can be per-
formed using the well-characterized/published
method as the reference method. The overall
accuracy of the method is determined as the total
number of samples yielding genotype calls iden-
tical to those determined by direct DNA
sequencing (or by using a well-characterized
method) and is expressed as percent accuracy.
Sample stability
While the reported stability of DNA is well
known, and the literature boasts a large number
of studies that have determined the effect of stor-
age time and temperature on both the quantity
(yield) and quality (A260:A280 ratio) of
genomic DNA in whole blood and isolated
DNA samples [25–27], very few validation studies
have been published regarding the stability of
certain sample types and/or genomic DNA with
respect to detecting accurate genotypes in the
stored samples. This is of greater interest for
sample matrices where degradation is known to
occur, but in order to formally document stabil-
ity it is more informative to establish the temper-
ature- and time-dependent stability of the tissue
of interest by using genotyping accuracy of the
isolated genomic DNA as the end point assess-
ment, rather than relying on the yield and appar-
ent quality of the isolated nucleic acid as the
indicator of DNA stability.
Validation samples & quality assessment
To demonstrate the ability of a designed assay to
detect wild-type, heterozygous polymorphic and
homozygous polymorphic alleles, samples with
preliminary data or annotation that suggests they
bear each allele combination should be included
in the initial panel of samples to be used for assay
validation if possible. We typically design an ini-
tial assay that exhibits acceptable in silico specific-
ity, and then screen several hundred samples to
identify genomic DNA (gDNA) samples from at
least 40 different individuals that will be
informative for analytical validation purposes.
For moderate- or high-frequency alleles, screen-
ing a relevant population of gDNA samples
typically results in the identification of at least
one or more samples for each allele combination.
For example, given that the CYP2D6*17
365
REVIEW – Isler, Vesterqvist & Burczynski
polymorphism has an allele frequency of 0.3% in
Caucasians, 34% in African–Americans and has
yet to be identified in Asians [28], a panel of Afri-
can–American gDNA should be screened to
identify samples heterozygous and homozygous
for the *17 polymorphism.
Panels of gDNA isolated from individuals of
a specified ethnicity or geographic location,
termed ‘variation panels’, are available from
suppliers such as Coriell Cell Repositories
(Camden, NJ, USA). Variation panels can be
screened using the genotyping assay in ques-
tion to initially identify polymorphic samples
to be included in the validation panel, since all
results generated using the genotyping assay
will ultimately be confirmed by either dideoxy
sequencing or by comparison with results
obtained by another assay based a different
technology. The Coriell Cell Repository
Pharmacogenetics Resource website lists
genomic DNA samples known to contain
polymorphisms in selected metabolic enzymes,
and these samples can be individually pur-
chased for inclusion in the validation sample
panel [105]. For low frequency alleles/mutations
(<1% prevalence), identification of samples
having the polymorphism of interest is some-
times not possible unless large numbers of
samples are screened.
Once the analytical validation of a geno-
typing assay is completed, approved and docu-
mented, the assay can be applied to the analysis
of clinical samples. Multiple QC samples are
included in each analytical run (typically a
96-well or 384-well format) to control for
method performance, including controls for
cross-contamination (water controls/non-PCR
amplified controls) and controls measuring the
ability of the assay to identify wild-type,
heterozygous and homozygous allele combina-
tions. Depending on the throughput of the
platform, QC samples represent approximately
10% of the samples analyzed in any given ana-
lytical run of clinical samples. To accept an
analytical run of clinical samples, all positive
QC samples (of both homozygous allele and
heterozygous allele combinations) must be
assigned the correct genotypes, and the nega-
tive control QC samples (water/non-PCR
amplified templates) must not give any geno-
typing results. QCs are subjected to the same
criteria used to determine acceptable genotyp-
ing calls in the clinical samples and if QC sam-
ples fail, the entire analytical run must be
repeated for the clinical samples in question.
366 Pharmacogenomics (2007) 8(4)
Expert commentary
The identification of an increasing number of
polymorphisms that may predict patient
responses and ultimately guide therapeutic
decision-making has brought to the forefront
the need to analytically validate targeted geno-
typing methods. While the technological com-
plexity of genotyping methods continues to
advance, the principles used to demonstrate the
analytical performance of these methods
remain the same. The performance criteria
described briefly herein are broadly applicable
to any sequence detection assay, despite our
focus on three PCR-based end point assays,
primer extension MALDI-TOF MS, Pyrose-
quencing and Taqman-based allelic discrimina-
tion. Specificity hails as perhaps the most
significant validation concept since it is one of
the more difficult parameters to demonstrate
and has a far-reaching impact on the accuracy
of genotype determinations.
Exploratory genotyping assays designed to
query SNPs in novel target genes during the
clinical development of a drug candidate may
or may not ultimately reach codiagnostic sta-
tus. In some cases they may simply guide
research and development of future thera-
peutics. Nonetheless, even for exploratory
biomarkers such as these, it is advantageous to
implement a process wherein novel genotyping
assays developed in house to query new SNPs
will be readily transferable to outside diagnostic
companies, should the need arise to assay these
SNPs in large-scale studies. Preparation of
detailed method procedures to allow for rapid
assay transfer, and assay validation reports that
require review and approval by a formalized
assay validation committee, can certainly
streamline and facilitate the development of
robust in-house genotyping methods that may
one day become molecular diagnostics.
Future perspective
Whole-genome association (WGA) studies are
becoming more routinely employed in the
search of candidate genetic loci causally related
to human disease, and the costs associated with
whole-genome analysis are likely to decrease in
the coming years. WGA studies typically query
hundreds of thousands of SNPs in a much
smaller number of samples. It is likely that tar-
geted genotyping assays designed to assess a
single or a small number of loci in larger num-
bers of confirmatory samples (e.g., procured
from large prospective studies, biobanks and
future science group
 Analytical validation of genotyping assays in the biomarker laboratory – REVIEW

repositories) will be increasingly employed to estimated and the real-time performance of an

rapidly determine whether polymorphisms ini-
tially identified in WGA studies appear truly
correlated with the phenotype of interest.
Technologies designed to accurately report
nucleotide sequences at defined positions in
gDNA continue to rapidly evolve. The three
technologies described in this review have
advantages and disadvantages; however,
analytical validation is only concerned with
establishing the performance characteristics of a
given analytical method. Thus, all present and
future technologies for genotyping will always be
amenable to analytical validation, such that the
expected performance of an assay can be
assay can be monitored with prudent use
of QCs.
Acknowledgements
The authors would like to thank Lynn Rutkowski for her sup-
port, and colleagues in Wyeth Discovery, Early Development
and Clinical Pharmacology, Translational Medicine, Clinical
Research and Development and World-Wide Regulatory
Affairs for their contributions to the efforts conducted by the
Wyeth Biomarker Laboratory in this field. We also thank all
the clinicians who have collected, and all of the subjects and
patients who have selflessly contributed peripheral blood sam-
ples in pharmacogenetic studies to further our understanding
of genetics, drug interactions and human disease.

Executive summary
• Whole-genome association (WGA) studies are identifying novel genetic markers, and the demand for reliable targeted
genotyping assays amenable to high-throughput analysis is expected to increase as more and more confirmatory studies are
required to validate initial findings in WGA studies.

• Recent guidance documents from the US FDA summarize aspects of both general pharmacogenomic biomarker validation and
analytical validation of pharmacogenomic assays, and provide excellent resources for laboratories developing pharmacogenomic
tests to support biomarker studies.

• Matrix-assisted laser desorption ionization time-of-flight mass spectrometry assays employ primer extension reactions in the
presence of mixtures of deoxynucleotides and dideoxynucleotides in order to determine the identity of the original nucleotide
juxtaposing the extension primer.

• Pyrosequencing® assays employ direct sequencing reactions using ordered nucleotide addition in the presence of luciferase-
related enzymes to determine the identity of nucleotides in the target gene as demonstrated by the generation of a luminescence
signal upon nucleotide addition.

• Allelic discrimination assays employ probe-based hybridization to determine the identity of the nucleotide present at a
polymorphic locus by measuring fluorescence of different fluorophores conjugated to oligonucleotide probes designed to
hybridize perfectly to either wild-type or mutant (polymorphic) sequences in the target gene.

• The possible presence of pseudogenes and/or regions of high homology elsewhere in the human genome sequence dictates that
careful in silico assessments of assay specificity should be performed before genotyping assays are tested in the
pharmacogenomic laboratory.

• While a number of different technologies have been designed to query nucleotide identity at exact locations in genomic DNA, all
of the technologies can be subjected to the principles of analytical validation to determine their suitability for assessing single
nucleotide polymorphsims in target genes.

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• Stability study looking at the effect of
various variables on DNA isolation.
27. Madisen L, Hoar DI, Holroyd CD,
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Websites
101. US FDA: Guidance for Industry.
Pharmacogenomic Data Submissions. FDA,
MD, USA (2005)
www.fda.gov/cder/guidance/6400fnl.pdf
• Landmark guidance document drafted to
facilitate the incorporation of personalized
medicine and pharmacogenomic principles
into drug development.
102. US FDA: Guidance for Industry and FDA
Staff. Class II Special Controls Guidance
Document: Drug Metabolizing Enzyme
Genotyping System. FDA, MD, USA
(2005)
www.fda.gov/cdrh/oivd/guidance/1551.pdf
• Helpful guidance document specifically
addressing the validation of genotyping
devices, with principles that are broadly
applicable to genotyping assays in general.
103. Oeth P, Beaulieu M, Park C et al.: iPLEX
Assay: Increased Plexing Efficiency and
Flexibility for MassARRAY® System
Through Single Base Primer Extension with
Mass Modified Terminators. Sequenom®
Inc., CA, USA (2005)
www.sequenom.com/Assets/pdfs/brochures/
IPLEX%20App%20Note%20External.pdf
104. The design process for a new generation of
quantitative gene expression analysis tools:
Taqman probe-based assays for human,
mouse and rat genes
www.appliedbiosystems.com/pebiodocs/001
13967.pdf
105. Coriell Institute for Medical Research:
National Institute of General Medical
Sciences Human Genetic Cell Repository
http://ccr.coriell.org/nigms/nigms_cgi
future science group